Activation of the P2RX7/IL-18 pathway in immune cells attenuates lung fibrosis

Serena Janho dit Hreich; Thierry Juhel; Sylvie Leroy; Alina Ghinet; Frederic Brau; Véronique Hofman; Paul Hofman; Valérie Vouret-Craviari

doi:10.7554/eLife.88138.1

eLife assessment

This study presents a potentially valuable discovery which indicates that activation of the P2RX7 pathway can reduce the degree of lung fibrosis caused by other inflammatory pathways. If confirmed, the study could clarify the role of specific immune networks in the establishment and progression of lung fibrosis. However, the presented data and analyses are incomplete as they rely on limited pharmacological treatments and because there is an absence of key control studies, validation experiments and statistical analyses.

https://doi.org/10.7554/eLife.88138.1.sa3

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Abstract

Idiopathic pulmonary fibrosis (IPF) is an aggressive interstitial lung disease associated with progressive and irreversible deterioration of respiratory functions that lacks curative therapies. Despite IPF being associated with a dysregulated immune response, current antifibrotics aim only at limiting fibroproliferation. We show here that the P2RX7/IL-18/IFNG axis is downregulated in IPF patients and that P2RX7 has immunoregulatory functions. Using our positive modulator of P2RX7, we show that activation of the P2RX7/IL-18 axis in immune cells limits lung fibrosis progression in a mouse model by favoring an anti-fibrotic immune environment, with notably an enhanced IL-18-dependent IFN-γ production by lung T cells leading to a decreased production of IL-17 and TGFβ. Overall, we show the ability of the immune system to limit lung fibrosis progression by targeting the immunomodulator P2RX7. Hence, treatment with a small activator of P2RX7 may represent a promising strategy for patients with lung fibrosis.

Introduction

Idiopathic pulmonary fibrosis (IPF) is an aggressive interstitial lung disease associated with progressive deterioration of respiratory function that is ultimately fatal. It is characterized by destruction of the lung architecture due to accumulation of fibroblasts and extracellular matrix proteins, resulting in increased lung stiffness and impaired normal breathing.

Pirfenidone and nintedanib have been FDA approved for the treatment of IPF since 2014. They target respectively the key fibrotic cytokine TGFβ and several receptor tyrosine kinases, thereby affecting fibroblast activation and extracellular matrix protein production (1). However, they only slow down the progression of the disease, so new therapeutic strategies and targets are needed.

Fibrosis is also associated with inflammation. In fact, fibrosis is a process of excessive wound healing and tissue remodeling due to repeated epithelial injuries releasing damage-associated molecular patterns (DAMPs) that trigger both the adaptive and innate immune systems. Although inflammation has not been considered a target in IPF, due to unsuccessful initial clinical trials of anti-inflammatory drugs (2) or cyclophosphamide during exacerbations (3), growing evidence suggest that altering specific immune populations that promote or attenuate disease progression may be beneficial (4).

Extracellular adenosine triphosphate (eATP) is a DAMP, released in high concentrations from injured cells in IPF patients (5). High levels of eATP are recognized by the P2X7 receptor (P2RX7) and are both required for the establishment of the bleomycin lung fibrosis mouse model (5). Activation of P2RX7 induces the opening of macropores, resulting in cell death (6), but also leads to the assembly of the NLRP3 inflammasome and the release of mature IL-1β and IL-18 (7). Consequently, P2RX7 has the ability to trigger an immune response.

IL-1β is a proinflammatory cytokine with high profibrotic properties, as it promotes collagen deposition through IL-17A and TGFβ production (8–10) but also promotes the activation and recruitment of inflammatory cells such as eosinophils and neutrophils. Indeed, deficiency of IL-1βR or its blockade ameliorate experimental fibrosis (11–13). In contrast, the role of IL-18 is not clear. Indeed, conflicting experimental studies show that IL-18 could either promote (14) or attenuate (15) fibrosis. However, high levels of IL-18BP, a natural antagonist of IL-18, are associated with reduced overall survival in IPF patients (16), suggesting that the activity of IL-18 may be required for improved survival.

IL-18 was originally described as IFN-γ-inducing factor (IGIF) and therefore boosts IFN-γ production by T cells and NK cells (17). Not only has IFN-γ antiproliferative properties (18) but it also inhibits TGFβ activity (19, 20) and therefore inhibits fibroblast activation and differentiation into myofibroblasts, alleviates TGFβ-mediated immunosuppression, inhibits extracellular matrix accumulation and collagen production (21–24) and thus promotes an antifibrotic immune microenvironment, making IFN-γ a cytokine with antifibrotic properties. However, parenteral systemic administration of IFN-γ failed in clinical trials (INSPIRE; NCT 00075998) (25), whereas local administration by inhalation showed promising results (26–30).

One way to increase IFN-γ production locally and selectively in the lung would be to alter the phenotype of T cells since the polarization of T lymphocytes has been shown to impact fibroblasts’ fate and immune infiltrate (4, 31). Indeed, T cells have been recently shown to selectively kill myofibroblasts through IFN-γ release and limit the progression of lung and liver fibrosis in preclinical models (32) and set up an immune memory in the long term since IFN-γ-producing tissue resident memory T cells protect against fibrosis progression (33), highlighting the importance IFN-γ producing T cells in this disease. Accordingly, CD4⁺-producing IFN-γ T cells are decreased in IPF and correlate with a better prognosis in IPF patients (31, 34, 35).

Given the ability of IL-18 to shape the phenotype of T cells by inducing IFN-γ, we proposed to increase local IFN-γ production via the P2RX7/IL-18 axis as a therapeutic strategy in pulmonary fibrosis. We used a P2RX7-specific positive modulator, developed in our laboratory, which has the particularity to only increase IL-18 levels in the presence of high eATP (36).

Results

Expression of P2RX7 and IL-18 activity are dampened in IPF patients

The canonical release of IL-18 is due to activation of the P2RX7/NLRP3 pathway (7). Since high levels of eATP are found in IPF patients (5) and P2RX7 is activated by such levels, it was of particular interest to investigate the involvement of P2RX7 in this disease. We used a publicly available dataset of lung homogenates from control and IPF patients (GSE47460) and compared the expression levels of P2RX7 and markers of fibrosis, namely ACTA2, COL1A2, COL3A1 and TGFB3. We found that the expression of P2RX7 is downregulated in IPF patients (Figure 1A and B), as well as the components of the NLRP3 inflammasome (Supplemental Figure 1). Since IL-18 is constitutively expressed (37), which partly explains the lack of difference between control and IPF patients (Figure 1B), we investigated the signaling pathway downstream of IL-18. IL-18 binds to its receptor IL-18R1 coupled to its adaptor protein IL-18RAP which is required for IL-18 signaling and IFN-γ expression. We showed that IL-18R1, IL-18RAP and IFN-γ (Figure 1B) are downregulated in IPF patients. Knowing that the modulation of the phenotype of T cells is promising in IPF (4), we checked whether P2RX7 and IL-18 are linked to an immune response in IPF using Gene Set Enrichment Analyses. Indeed, we showed that the expression of P2RX7 and IL-18 signaling (IL-18 and IL-18RAP) correlates with the IFN-γ response as well as with immunoregulatory interactions required for changing the phenotype of T cells (Figure 1C, Supplemental Figure 1B). These results highlight that the P2RX7/IL-18 signaling pathway is dampened and that this pathway is able to modulate the immune response in IPF patients.

The P2RX7/IL-18/IFN-γ pathway is downregulated in IPF
**(A)** Heatmap of mRNA expression of P2RX7 in control and IPF patients with a cluster of fibrosis-associated genes. Raw p-values are shown (Limma). **(B)** mRNA expression of *P2RX7, IL18, IL18R1, IL18RAP* and *IFNG* between control and IPF patients from 213 individuals, corresponding to 91 controls and 122 IPF patients. Two-tailed unpaired t-test with Welch’s correction, ***p < 0.001, ****p < 0.0001. **(C)** Gene set enrichment analysis (GSEA) plot associating P2RX7 mRNA levels from IPF patients with three immunological signatures. The green line represents the enrichment score and the black lines the specific signature-associated genes. NES: Normalized enrichment score, FDR: False discovery rate. Pearson’s correlation test.

Activation of P2RX7 inhibits the onset of pulmonary fibrosis in the bleomycin mouse model

We therefore hypothesized that activation of the P2RX7/IL-18 signaling may restrain lung fibrosis progression. To test this hypothesis, we decided to boost the P2RX7/IL-18 signaling in the bleomycin (BLM)-induced lung fibrosis mouse model. To do so, we used a positive modulator of P2RX7, called HEI3090, which enhances P2RX7’s activity only in the presence of high eATP levels (36). Indeed, knowing that high eATP levels are found in IPF patients as well as in this mouse model of pulmonary fibrosis (5), we thought that HEI3090 will selectively enhance the activity of P2RX7 in the lungs.

We first tested the antifibrotic potential of HEI3090 on mice having an established fibrosis (Figure 2A). Activation of P2RX7 with HEI3090 in mice 7 days after BLM administration reduced the development of pulmonary fibrosis, as evidenced by less thickening of alveolar walls and free air space (Figure 2B). Fibrosis severity was evaluated using the Ashcroft score. To overcome the heterogeneity of fibrosis within the lobes, we scored the whole surface of the lung, and the result represents the mean of each field (Figure 2C and supplementary Figure 2). As accumulation of extracellular matrix proteins is a hallmark of fibrosis, we also checked collagen levels in the lungs of vehicle and HEI3090-treated mice by measuring Sirius Red polarized light images of the entire lung. We showed that collagen content was reduced in lungs of HEI3090-treated mice (Figure 2, B and D). We also tested the ability of HEI3090 to limit lung fibrosis progression when administered during the inflammatory phase of the BLM model (Figure 2E) that mimics the exacerbation episodes in IPF patients (38). HEI3090 was also able to inhibit the onset of lung fibrosis in this setting given the reduced fibrosis score (Figure 2, F and J) and collagen content in lungs of HEI3090-treated mice (Figure 2, F and H). These results show that activation of P2RX7 with HEI3090 inhibits the lung fibrosis progression and is effective during both the fibroproliferative and acute inflammation phase of the BLM-induced pulmonary fibrosis mouse model.

Activation of P2RX7 with HEI3090 inhibits lung fibrosis progression
**(A)** Experimental design. WT mice were given 2.5 U/kg of bleomycin by i.n. route. At the end of the inflammatory phase, 1.5 mg/kg of HEI3090 or vehicle were given daily until day 21. **(B)** Representative images of lung sections at day 21 after treatment stained with H&E and Sirius Red, Scale bar= 100 µm. **(C)** Fibrosis score assessed by the Ashcroft method. **(D)** Collagen levels in whole lung of mice assessed on Sirius Red-polarized images. **(E)** Experimental design. WT mice were given 2.5 U/kg of bleomycin by i.n. route. 1.5 mg/kg of HEI3090 or vehicle were given daily until day 14. **(F)** Representative images of lung sections at day 14 after treatment stained with H&E and Sirius Red, Scale bar= 100 µm. **(G)** Fibrosis score assessed by the Ashcroft method. **(H)** Collagen levels in whole lung of mice assessed on Sirius Red-polarized images. Each point represents one mouse, two-tailed Mann-Whitney test, p values: *p < 0.05, **p < 0.01. WT: Wildtype, BLM: bleomycin, i.p.: intraperitoneal, i.n.: intranasal, H&E: hematoxylin & eosin, AU: arbitrary units.

HEI3090 shapes immune cell infiltration in the lungs

As P2RX7 has immunoregulatory functions in IPF (Figure 1) and as HEI3090 has antifibrotic activity (Figure 2), we next investigated if HEI3090 had an impact on both the immune landscape of the lung and production of cytokines. We show that lung CD3+ T cells were more biased to produce IFN-γ than the profibrotic IL-17A cytokine when mice were treated with HEI3090 (Figure 3A). This result is consistent with what we have shown in IPF patients (Figure 1). This biased production of IFN-γ is only seen in CD3+ T cells and not in overall lung immune cells (Supplemental Figure 3A) nor in the subsets of T lymphocytes (Figure 3B, Supplemental Figure 3A) or NK cells (Supplemental Figure 3B). Although levels of CD3+ T cells and T cell subsets were unchanged (Supplemental Figure 3C), including the profibrotic Th17 cells (Figure 3D), IL-17A production by Th17 cells is markedly attenuated after HEI3090 treatment (Figure 3D), consistent with the ability of IFN-γ to inhibit IL-17A production (39). Considering the strong profibrotic properties of TGFβ and its mutual antagonism with IFN-γ (40, 41), we checked whether HEI3090 had an effect on TGFβ levels. Indeed, treatment with HEI3090 reduced TGFβ-producing immune cells in the lung as well as TGFβ production (Figure 3E). Notably, HEI3090 treatment reduced TGFβ production in NK cells but not in T-cell subsets (Supplemental Figure 3E).

HEI3090 favors an anti-fibrotic immune signature in the lungs
WT mice were given 2.5 U/kg of bleomycin by i.n. route and treated daily i.p. with 1.5 mg/kg of HEI3090 or Vehicle. Lungs were analyzed by flow cytometry at day 14. **(A)** Contour plot of IFN-γ and IL-17A producing T cells (CD3⁺NK1.1^-) (left) and ratio of IFN-γ over IL-17A in T cells (CD3⁺NK1.1^-) (right). **(B)** Percentage of IFN-γ producing CD4⁺ and CD8⁺ T cells. **(C)** Percentage and GMFI of IL-17A⁺ cells of CD4⁺ T cells (CD3⁺CD4⁺NK1.1^-). **(D)** Percentage and GMFI of TGFβ in CD45⁺ cells. **(E)** Dotplot showing lung inflammatory monocytes, gated on lineage^-CD11c^-CD11b⁺ cells (left) and percentage of lung inflammatory monocytes (Ly6C^highLy6G^-) (right) **(F)** Percentage of alveolar macrophages (CD11c⁺SiglecF⁺) and **(G)** lung eosinophils (CD11b⁺SiglecF⁺CD11c^-). Each point represents one mouse, data represented as violin plots or mean±SEM, two-tailed Mann-Whitney test, *p < 0.05, **p < 0.01. GMFI: geometric mean fluorescence intensity. i.n.: intranasal, i.p.: intraperitoneal.

Pulmonary fibrosis is also favored and driven by the recruitment of inflammatory cells, mainly from the myeloid lineage. Monocytes are highly inflammatory cells that are recruited to the lung and differentiate into alveolar macrophages, both of which have strong profibrotic properties (42–44). We demonstrated that in HEI3090-treated mice, the number of inflammatory monocytes decreased markedly (Figure 3E), whereas the number of alveolar macrophages remained unchanged (Figure 3F), consistent with the prognostic ability of monocyte count in IPF progression (45–48). We also examined other inflammatory cells with profibrotic properties, such as eosinophils (49) that are less present in HEI3090-treated lungs (Figure 3G), or PMN levels that remain unchanged by HEI3090 treatment (Supplemental Figure 3C).

We also wondered if the activation of P2RX7 had a systemic effect by analyzing immune changes in mice’s spleens. No significant change in cell populations was observed in the spleens of mice (Supplemental Figure 4B) when HEI3090 was administrated in the early phase of the BLM model suggesting a local lung activity of the molecule. However, HEI3090 reactivated a systemic immune response with higher levels of dendritic cells and lymphocytes in the spleens of mice treated during the fibroproliferation phase (Supplemental Figure 4D). These results show the ability of HEI3090 to shape the immune response locally and impact the progression of fibrosis systemically even in the fibroproliferative phase of the BLM model.

Altogether these results demonstrate that activation of P2RX7 with HEI3090 promotes an antifibrotic cytokinic profile in lung immune cells and attenuates lung inflammation.

HEI3090 requires the P2RX7/NLRP3/IL-18 pathway in immune cells to inhibit lung fibrosis

We wanted to further investigate the mechanism of action of HEI3090 by identifying the cellular compartment and signaling pathway required for its activity. Since the expression of P2RX7 and the P2RX7-dependent release of IL-18 are mostly associated with immune cells(50), and since HEI3090 shapes the lung immune landscape (Figure 3), we investigated whether immune cells were required for the antifibrotic effect of HEI3090.

To do so, we performed an adoptive transfer experiment with WT P2RX7-expressing splenocytes (Figure 4A, supplemental Figure 5E) into p2rx7^-/- mice one day before BLM administration. We show that restriction of P2RX7 expression on immune cells restored the antifibrotic effect of HEI3090 based on the architecture of the lung, with lungs of HEI3090-treated mice showing more free airspace and thinner alveolar walls (Figure 4B), as well as an overall lower fibrosis score (Figure 4C) than control lungs. Since the bleomycin mouse model relies on P2RX7-expressing epithelial cells (5), we wanted to validate further the role of P2RX7-expressing immune cells in a mouse model where P2RX7 is expressed by non-immune cells. To do so, we reduced both the expression of P2RX7 and its activity by repeated i.v. administration of p2rx7^-/- splenocytes in WT mice (Supplemental Figure 5). In this setting, HEI3090 was unable to limit the progression of fibrosis. Moreover, we show that the activity of HEI3090 requires P2RX7 expression, as this effect was lost in p2rx7^-/- mice (Supplemental Figure 6, A and B) (36). These results highlight the important role of immune cells and rules out a major role of non-immune P2RX7-expressing cells, such as fibroblasts, in the antifibrotic effect of HEI3090.

The P2RX7/NLRP3/IL-18 pathway in immune cells is required for HEI3090’s antifibrotic effect
**(A)** Experimental design. p2rx7-/-mice were given 3.106 WT, nlrp3-/-or il18-/-splenocytes i.v. one day prior to BLM delivery (i.n. 2.5 U/kg). Mice were treated daily i.p. with 1.5 mg/kg HEI3090 or vehicle for 14 days. **(B,D,F)** Representative images of lung sections at day 14 after treatment stained with H&E and Sirius Red, scale bar= 100 µm. Fibrosis score assessed by the Ashcroft method of adoptive transfer of WT splenocytes, **(E)** nlrp3-/-splenocytes and **(G)** il18-/-splenocytes. **(H)** IL-18 levels in sera of WT BLM-induced mice at day 14 **(I)** Ratio of IFN-γ over IL-17A in lung T cells (CD3+NK1.1-) of WT mice neutralized for IL-18 or not (isotype control) every 3 days. Each point represents one mouse, two-tailed Mann-Whitney test, *p < 0.05. WT: Wildtype, BLM: bleomycin, i.p.: intraperitoneal, i.n.: intranasal, i.v.: intravenous, H&E: hematoxylin & eosin.

To test the importance of the NLRP3/IL-18 pathway downstream of P2RX7, we performed an adoptive transfer of nlrp3^-/- and il18^-/- splenocytes into p2rx7^-/-mice, expressing similar levels of P2RX7 as WT splenocytes (Supplemental Figure 6E), but also the same levels of IL-18 and NLRP3 (Supplemental Figure 6F). The absence of NLRP3 and IL-18 in P2RX7-expressing immune cells abrogated the ability of HEI3090 to inhibit lung fibrosis because the lung architecture resembled that of control mice (Figure 4, D-G). Consistent with the requirement of IL-18 for HEI3090’s antifibrotic activity, activation of P2RX7 with this molecule in WT mice increased the levels of IL-18 in the sera of these mice compared to control mice (Figure 4H). Moreover, neutralization of IL-18 abrogated the increase of the IFN-γ/IL-17A ratio by lung T cells (Figure 4I), highlighting furthermore the necessity of IL-18 for the antifibrotic effect of HEI3090.

Not only does the activation of the P2RX7/NLRP3 pathway leads to the release of IL-18, but also induce the release of the pro-inflammatory and pro-fibrotic IL-1β cytokine. However, IL-1β was not involved in the antifibrotic effect of HEI3090 (Supplemental Figure 6C), nor were its levels affected by HEI3090 in WT mice (Supplemental Figure 6D).

Overall, we show that the P2RX7/NLRP3/IL-18 axis in immune cells is required to limit lung fibrosis progression, highlighting the efficacy in targeting the immune system in this disease.

Discussion

A major unmet need in the field of IPF is new treatment to fight this uncurable disease. In this study, we demonstrate the ability of immune cells to limit lung fibrosis progression. Based on the hypothesis that a local activation of a T cell immune response and upregulation of IFN-γ production has antifibrotic proprieties, we used the HEI3090 positive modulator of the purinergic receptor P2RX7, previously developed in our laboratory (36), to demonstrate that activation of the P2RX7/IL-18 pathway inhibits lung fibrosis in the bleomycin mouse model.

We have demonstrated that lung fibrosis progression is inhibited by HEI3090 in the fibrotic phase but also in the acute phase of the BLM fibrosis mouse model, i.e. during the period of inflammation. This lung fibrosis mouse model is classically used in preclinical studies and has been designated recently as the best model for IPF (51). The efficacy of HEI3090 to inhibit lung fibrosis was evaluated histologically on the whole lung’s surface by evaluating the severity of fibrosis and collagen levels using respectively the Ashcroft score (52) and polarized-light microscopy of Sirius Red staining to visualize collagen fibers in the whole lung. In both settings, HEI3090 reduced alveolar wall thickness and accumulation of collagen fibers in the entire lung, highlighting a comprehensive pre-clinical evaluation of HEI3090 as a new anti-fibrotic therapy.

Our study showed that inhibition of fibrosis progression by HEI3090 was associated with an increased production of IFN-γ by lung T cells that was dependent on an increased release of IL-18. We also showed that expression of the P2RX7/IL-18/IFN-γ pathway is attenuated in IPF patients where TGFβ levels are high (52), consistent with the ability of TGFβ to downregulate IL-18R expression and IL-18-mediated IFN-γ production (53). These results confirm the beneficial effects of enhancing activation of the P2RX7/IL-18/IFN-γ pathway.

P2RX7 is expressed by various immune and non-immune cells, but its expression is the highest in dendritic cells (DCs) and macrophages (54), from which IL-18 is mainly released to shape the T-cell response (55) and increase T-cell IFN-γ production (56). Collectively, and consistent with the immunomodulatory properties of P2RX7, these observations suggest that HEI3090 may target P2RX7-expressing antigen-presenting cells to influence the T-cell response, which may explain the selective T-cell increase in IFN-γ in HEI3090-treated mice. Accordingly, we have previously shown that HEI3090 targets the P2RX7/IL-18 axis in DCs to shape the immune response in a lung tumor mouse model (36).

Activation of P2RX7 with HEI3090 not only increased IFN-γ production by T cells but it also reshaped the immune and cytokinic composition of the lung. Indeed, lungs of HEI3090-treated mice show a decrease in IL-17A production by Th17 cells and TGFβ production by lung immune cells. Moreover, lung inflammation is dampened after HEI3090 treatment, since the number of inflammatory monocytes and eosinophils decreases. It is not known whether this cytokinic and anti-inflammatory switch is solely due to the IL-17A and TGFβ-suppressive property of IFN-γ (57–59), or whether it is a combination with the cell death-inducing property of P2RX7 (6).

The novelty of this approach is that it targets and alters the immune environment of the lung. Indeed, the use of a P2RX7-specific modulator that acts only in an ATP-rich environment was effective in promoting an anti-inflammatory and anti-fibrotic phenotype by altering several key mediators of the disease. In contrast to current therapies (60), no side effects were observed when mice were treated with HEI3090, further supporting this targeted approach. Furthermore, since current therapies (pirfenidone or nintedanib) and HEI3090 have different mechanisms of action, the combination of these therapies could have additive or synergistic effects.

It is also important to note that our strategy is unconventional, as P2RX7 is known to be pro-inflammatory through IL-1β-release. However, HEI3090 was unable to increase IL-1β release in vivo in this model, even though it efficiently increased IL-18 release. This observation is consistent with our previous in vivo and in vitro studies (36), allowing us to rule out the pro-inflammatory and pro-fibrotic effects of P2RX7-dependent IL-1β release. Consistent with this finding, it has been reported that ATP stimulation of alveolar macrophages derived from both IPF and lung cancer patients resulted only in an increase in IL-18 (61, 62), which was explained by an impaired NLRP3 inflammasome and a defective autophagy described in IPF patients (63). Interestingly, autophagy can be regulated by P2RX7 (64) and is one of the pathways that allow the release of IL-1β from the cell (65). Moreover, unlike IL-1β, IL-18 is constitutively expressed in human and mouse immune cells (37) but also in non-immune cells such as fibroblasts (66) and lung epithelium (67) and is both matured and released after NLRP3 activation. Therefore, it is currently not known whether the lack of IL-1β release is due to the different cytokine expression pattern or whether there is IL-1β-specific regulation following an enhanced activation of P2RX7 or a defective NLRP3 inflammasome.

In this study, we emphasize the importance of IL-18 for an antifibrotic effect. Several studies have indicated that P2RX7/NLRP3/IL-18 promote disease progression using knock out mice or inhibitors (5, 14, 66). However, experimental mouse models rely on lung epithelial cell injury that has been shown to activate the NLRP3 inflammasome in the lung epithelium as a first step (5, 12) and release danger signals that activate the immune system as a second step. Therefore, initial lung injury to epithelial cells is reduced or absent in p2rx7^-/- and nlrp3^-/- mice, indicating that P2RX7 and NLRP3 are required for the establishment of the bleomycin mouse model rather than their role in an already established fibrosis, which has not yet been studied. In addition, NLRP3 and the release of IL-1β and IL-18 from fibroblasts have been shown to promote myofibroblast differentiation and extracellular matrix production (66, 68, 69). These observations suggest that fibrosis mouse models initially rely on NLRP3 activation by nonimmune cells and encourage further studies on the contribution of the NLRP3 inflammasome in immune cells to fibrosis progression in vivo. Since we have highlighted the importance of this pathway in immune cells for delaying fibrosis progression, we propose that IL-18 may have different effects depending on the cell type.

Beside an urgent need of new treatments for IPF, there is also a lack of biomarkers, such as prognostic biomarkers, markers of disease activity, or markers of drug efficacy. Our results suggest the possible benefit of an active IL-18 in the pathophysiology of pulmonary fibrosis and warrant analysis of IL-18 as a promising biomarker for predicting outcome in IPF patients. Given the potential effects of pirfenidone and nintedanib on IL-18 levels in preclinical models (70–72), determining IL-18 shifts during treatment would be highly interesting to evaluate potential changes in patients’ outcome and to examine IL-18 levels which may be helpful in the long run for patient treatment strategy and subsequent introduction of pipeline drugs (73).

Overall, we highlight in this study the ability of the P2RX7/NLRP3/IL-18 pathway in immune cells to inhibit the onset of lung fibrosis by using a positive modulator of P2RX7 that acts selectively in an eATP-rich environment such as fibrotic lung. The unique feature of this strategy is that it enhances the antifibrotic and it attenuates the pro-fibrotic properties of immune cells, with no reported side effects.

Methods

Microarray

mRNA expression profile was obtained from Gene Expression Omnibus (GEO) database (GSE47460) using the GPL14550. Microarray was done on whole lung homogenate from subjects undergoing thoracic surgery from healthy subjects with no lung-related pathology or from subjects diagnosed as having interstitial lung disease as determined by clinical history, CT scan, and surgical pathology. Expression profile belong to the Lung Tissue Research Consortium (LTRC). 122 patients with UIP/IPF and 91 healthy controls were analyzed in this study.

Mice

Mice were housed under standardized light–dark cycles in a temperature-controlled air-conditioned environment under specific pathogen-free conditions at IRCAN, Nice, France, with free access to food and water. All mouse studies were approved by the committee for Research and Ethics of the local authorities (CIEPAL #598, protocol number APAFIS 21052-2019060610506376) and followed the European directive 2010/63/UE, in agreement with the ARRIVE guidelines. Experiments were performed in accord with animal protection representative at IRCAN. p2rx7^−/− (B6.129P2-P2rx7tm1Gab/J, from the Jackson Laboratory) were backcrossed with C57BL/6J OlaHsD mice. C57BL/6J OlaHsD male mice (WT) were supplied from Envigo (Gannat, France).

Induction of lung fibrosis

WT or p2rx7^-/- male mice (8 weeks) were anesthetized with ketamine (25mg/kg) and xylazine (2.5 mg/kg) under light isoflurane and were given 2.5 U/kg of bleomycin sulfate (Sigma-Aldrich) by intranasal route. Mice were treated i.p. every day with vehicle (PBS, 10% DMSO) or with HEI3090 (1.5 mg/kg in PBS, 10% DMSO) (36) starting D1 or D7 post bleomycin delivery, as mentioned in the figures. After 14 days of treatment, lungs were either fixed for paraffin embedding or weighted and analyzed by flow cytometry. When mentioned, 200 µg of anti-IL-18 neutralizing antibody (BioXcell) or isotype control (IgG2a, BioXcell) were given by i.p. every three days starting one day prior to BLM administration.

Adoptive transfer in p2rx7 deficient mice

Spleens from C57BL/6J male mice (8-10 weeks) were collected and digested with the spleen dissociation kit (Miltenyi Biotech) according to the supplier’s instructions. 3.10⁶ splenocytes were injected i.v. in p2rx7^-/-mice 1 day before intranasal delivery of bleomycin. Mice were treated i.p. every day for 14 days with vehicle (PBS, 10% DMSO) or with HEI3090 (1.5 mg/kg in PBS, 10% DMSO). Nlrp3^-/- spleens were a kind gift from Dr Laurent Boyer, il18^-/- spleens from Dr George Birchenough and il1b^-/- spleens from Dr Bernhard Ryffel, all on a C57BL/6J background.

Histology

Lungs were collected and fixed in 3% formamide for 16 h prior inclusion in paraffin. Lungs sections (3 µm) were stained with hematoxylin & eosin or with Sirius red (Abcam) according to the supplier’s instructions. The severity of fibrosis was assessed on whole lungs using the Ashcroft modified method (74). The fibrosis score represents the mean of fields of 0.883 mm² each covering all the lobes of the lungs as shown in Supplementary Figure 2.

Levels of collagen on whole lungs were assessed on Sirius Red polarized light images of the entire lung taken with HD-Axio Observer Z1 Microscope ZEISS microscope using ImageJ. The collagen amount given by the polarization intensity of the Sirius red staining of the lung slices was quantified with a homemade ImageJ/Fiji (75) macro program. The mean gray value of the collagen staining was measured in the fibrotic regions excluding the signal coming from vessels and lung epithelia using dedicated masks. The binary masks were obtained after median filtering and manual thresholding, from the transmission images for the fibrotic one and the polarization images for the vessels. The intersection of these masks is then applied on the polarization image to get specifically the mean gray value of fibrotic collagen.

Flow cytometry and antibodies

Lungs or spleens were collected and digested with the lung or spleen dissociation kit (Miltenyi Biotech) according to the supplier’s instructions. Red blood cells were lysed using ACK lysis buffer (Gibco). Fc receptors were blocked using anti-CD16/32 (2.4G2) antibodies followed by surface staining by incubating cells on ice, for 20 min, with saturating concentrations of labeled Abs (Table 1) in PBS, 5% FBS and 0.5% EDTA. Tregs were identified using the transcription factor staining Buffer Set (eBioscience) for FoxP3 staining. Intracellular staining was performed after stimulation of single-cell suspensions with Phorbol 12-myristate 13-acetate (PMA at 50 ng mL⁻¹, Sigma), ionomycin (0.5 μg mL⁻¹, Sigma) and 1 μL mL⁻¹ Golgi Plug™ (BD Biosciences) for 4 h at 37°C 5% CO2. Cells were incubated with Live/Dead stain (Invitrogen), according to the manufacturer protocol prior to surface staining. Intracellular staining was performed using Cytofix/Cytoperm™ kit (BD biosciences) following the manufacturer’s instructions. Samples were acquired on CytoFLEX LX (Beckman Coulter) and analyzed using FlowJo (LLC).

ELISA

Sera of mice were collected at the end of the experiment and stored at −80 °C before cytokine detection by ELISA using mouse IL-1 beta/IL-1F2 (R&D) and IL-18 (MBL) according to the supplier’s instructions.

Western Blot

Single cell suspensions of whole lungs were lysed with Laemmli buffer (10% glycerol, 3% SDS, 10 mM Na2HPO4) with protease inhibitor cocktail (Roche). Proteins were separated on a 12% SDS-PAGE gel and electro transferred onto PVDF membranes, which were blocked for 30 min at RT with 3% bovine serum albumin or 5% milk. Membranes were incubated with primary antibodies (see Table 1) diluted at 4 °C overnight. Secondary antibodies (Promega) were incubated for 1 h at RT. Immunoblot detection was achieved by exposure with a chemiluminescence imaging system (PXI Syngene, Ozyme) after membrane incubation with ECL (Immobilon Western, Millipore). The bands intensity values were normalized to that of β-actin using ImageJ software.

Statistical analyses

All analyses were carried out using Prism software (GraphPad). Mouse experiments were performed on at least n = 5 individuals, as indicated. Mice were equally divided for treatments and controls. Data is represented as mean values and error bars represent SEM. Two-tailed Mann–Whitney and unpaired t-test were used to evaluate the statistical significance between groups. For survival analyses, the log-rank Mantel-Cox test was used. For correlation analyses, Spearman test was used for the Gene Set Enrichment Analyses (GSEA).

Data and materials availability: All data are available in the main text or the supplementary materials. RNAseq data from IPF and control patients were retrieved from GEO database under the accession number GSE47460.

Author contributions

Conceptualization and design: SJH, VV-C

Methodology: SJH, FB

Investigation: SJH, TJ, VV-C

Analysis and Interpretation: SJH, SL, PH, VH, VV-C

Reagents: AG

Writing: SJH, VV-C

Acknowledgements

The authors wish to thank Dr George Birchenough, Dr Laurent Boyer and Dr Bernhard Ryffel for sharing il18^-/-, nlrp3^-/- and il1b^-/- spleens respectively. This publication is based upon discussion from PRESTO COST action CA21130 supported by COST (European Cooperation in Science and Technology).

Funding: Ligue Nationale Contre le Cancer (SJH), Fondation pour la recherche médicale grant number #FDT202106013099 (SJH), ARC grant number ARCTHEM2021020003478 (SJH, VV-C), Cancéropôle PACA (VV-C)

The French Government (National Research Agency, ANR through the “Investments for the Future”: program reference #ANR-11-LABX-0028-01 (PH), Executive Unit for Financing Higher Education, Research, Development and Innovation (UEFISCDI), Bucharest, Romania (grant number PN-III-P4-ID-PCE-2020-0818, acronym REPAIR) (AG).

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Article and author information

Author information

Serena Janho dit Hreich
Université Côte d’Azur, CNRS, INSERM, IRCAN, 06108 Nice, France, FHU OncoAge, Nice, France
Thierry Juhel
Université Côte d’Azur, CNRS, INSERM, IRCAN, 06108 Nice, France
Sylvie Leroy
FHU OncoAge, Nice, France, Université Côte d’Azur, CNRS, Institut Pharmacologie Moléculaire et Cellulaire, Sophia-Antipolis, France, Université Côte d’Azur, Centre Hospitalier Universitaire de Nice, Pneumology Department, Nice, France
Alina Ghinet
Inserm U995, LIRIC, Université de Lille, CHRU de Lille, Faculté de médecine – Pôle recherche, Place Verdun, F-59045 Lille Cedex, France, Hautes Etudes d’Ingénieur (HEI), JUNIA Hauts-de-France, UCLille, Laboratoire de chimie durable et santé, 13 rue de Toul, F-59046 Lille, France, ‘Al. I. Cuza’ University of Iasi, Faculty of Chemistry, Bd. Carol I, nr. 11, 700506 Iasi, Romania
Frederic Brau
Université Côte d’Azur, CNRS, Institut Pharmacologie Moléculaire et Cellulaire, Sophia-Antipolis, France
Véronique Hofman
Université Côte d’Azur, CNRS, INSERM, IRCAN, 06108 Nice, France, FHU OncoAge, Nice, France, Laboratory of Clinical and Experimental Pathology and Biobank, Pasteur Hospital, Nice, France, Hospital-Related Biobank (BB-0033-00025), Pasteur Hospital, Nice, France
Paul Hofman
Université Côte d’Azur, CNRS, INSERM, IRCAN, 06108 Nice, France, FHU OncoAge, Nice, France, Laboratory of Clinical and Experimental Pathology and Biobank, Pasteur Hospital, Nice, France, Hospital-Related Biobank (BB-0033-00025), Pasteur Hospital, Nice, France
Valérie Vouret-Craviari
Université Côte d’Azur, CNRS, INSERM, IRCAN, 06108 Nice, France, FHU OncoAge, Nice, France
- Corresponding author: Valérie VOURET-CRAVIARI, IRCAN, 28 Avenue de Valombrose, 06108 Nice, France. Tel 33 (0)492031223. Email: valerie.vouret@univ-cotedazur.fr

Author Notes

Conflict of interest statement: All other authors declare they have no competing interests.

Version history

Sent for peer review: April 5, 2023
Preprint posted: April 10, 2023
Reviewed Preprint version 1: June 6, 2023
Reviewed Preprint version 2: December 4, 2023
Reviewed Preprint version 3: January 12, 2024
Version of Record published: February 1, 2024

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